The conflict between bacteria and their viruses – phages – is of eminent importance. Phages can threaten cultures of beneficial bacteria in the food industry or present an alternative to antibiotics by battling multidrug-resistant pathogens, known as phage therapy. Thus, bacterial phage defence systems, providing immunity against phages, are under intense research. Here, we present the toxin-antitoxin systems that co-opted the ubiquitous NAD+ molecule to ADP-ribosylate DNA for phage defence.

Within legume root nodules, rhizobia differentiate into bacteroids, which reduce N2 into NH3 for secretion to the plant. Bacteroids may be swollen and terminally differentiated or non-swollen and can regenerate outside nodules. It is unclear why these different endosymbiotic lifestyles exist and whether they differ in symbiotic efficiency. Here, we compared N2 fixing bacteroids of the near isogenic strains Rhizobium leguminosarum bv. phaseoli 4292 (Rlp4292) and R. leguminosarum bv. viciae A34 (RlvA34), nodulating Phaseolus vulgaris (common bean) and Pisum sativum (pea), respectively. The larger bean plants fixed more N2, but peas fixed 1.6-3-fold more per unit nodule mass. Values per unit volume were similar between bean and pea because bean nodules are 2.7-fold denser (i.e., mass per unit volume). Bean nodules have higher numbers of smaller (∼1/5 the volume) bacteroids than peas. Bean bacteroids are denser (i.e., 2.5-fold protein per unit volume) although less closely packed than pea bacteroids (i.e. more space between bean bacteroids). Critically, pea bacteroids, fix N2 at higher rates versus bean per unit bacteroid protein, as protein expression is skewed towards N2 fixation and TCA-cycle enzymes. Pea bacteroids infect 1.6 times the percentage of nodule volume of beans (i.e., 14.2% versus 9.1%). Overall, the increased packing density of pea bacteroids, as well as the bias of their proteome to nitrogenase, associated N2 fixation processes, and dicarboxylate metabolism, contributes to their greater symbiotic efficiency, which is likely driven by plant antimicrobial peptides.

Cereal root microbiomes harbour diverse diazotrophic bacteria, yet the taxa capable of sustained nitrogen fixation in association with cereal roots remain poorly characterized. Here, two high-performing nitrogen-fixing strains (B6 and J2) were isolated from barley roots and identified as belonging to the family Rhizobiaceae in the genus Paenirhizobium. Both strains possess plasmid-encoded canonical rhizobial nif and fix genes for nitrogen fixation but lack nodulation genes. Their genomes have a 5.7 Mb chromosome and four repABC plasmids. Unlike most nodulating rhizobia, strains B6 and J2 fixed nitrogen in laboratory culture on a range of carbon sources, achieving maximal activity on organic acids at low ammonium (<0.5 mM) and oxygen concentrations (1-3%). Both strains colonized the total root systems of barley plants, with population densities of 106 CFU g-1 fresh root weight. Strains fixed high levels of nitrogen on barley plants, similar to or greater than other known free-living diazotrophs. These findings expand the ecological context of rhizobial nitrogen fixation and identify cereal-associated Paenirhizobium as a previously unrecognized component of the diazotrophic cereal root microbiome.

Peptidoglycan is an essential component of the bacterial cell envelope-a mesh-like macromolecule that protects the bacterium from osmotic stress and its internal turgor pressure. The composition and architecture of peptidoglycan is heterogeneous and changes as bacteria grow, divide, and respond to their environment. Though peptidoglycan has long been studied via LC-MS/MS, the analysis of this data remains challenging as peptidoglycan's unusual composition and branching can't be handled by proteomics software. Here we describe user-friendly open-source tools and a web interface for building peptidoglycan databases, performing MS searches, and predicting the MS/MS fragmentation of muropeptides. We then use Rhizobium leguminosarum to describe a step-by-step strategy for the high-resolution analysis of peptidoglycan. The unprecedented detail of R. leguminosarum's peptidoglycan composition (>250 muropeptides) reveals even the subtlest remodelling between growth conditions. These new and easier to use tools enable more systematic analyses of peptidoglycan dynamics.

Background After two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom. Results To address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots. Conclusions DFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly.

Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate, the reason for the lack of N 2 fixation in the mutant has remained unclear. During our investigations into the role of AatA, we found that it catalyses an additional transamination reaction between aspartate and pyruvate, forming alanine. This secondary reaction runs at around 60 % of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate–pyruvate transaminase activity in R. leguminosarum , which is common in eukaryotic and many prokaryotic genomes. However, the aspartate-to-pyruvate transaminase reaction is not needed for N 2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N 2 fixation, rather than biosynthetic transamination to form an amino acid. Hence, the enzyme aspartase, which catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N 2 fixation in pea nodules.

Bacterial persistence in the rhizosphere and colonization of root niches are critical for the establishment of many beneficial plant-bacteria interactions including those between Rhizobium leguminosarum and its host legumes. Despite this, most studies on R. leguminosarum have focused on its symbiotic lifestyle as an endosymbiont in root nodules. Here, we use random barcode transposon sequencing to assay gene contributions of R. leguminosarum during competitive growth in the rhizosphere and colonization of various plant species. This facilitated the identification of 189 genes commonly required for growth in diverse plant rhizospheres, mutation of 111 of which also affected subsequent root colonization (rhizosphere progressive), and a further 119 genes necessary for colonization. Common determinants reveal a need to synthesize essential compounds (amino acids, ribonucleotides, and cofactors), adapt metabolic function, respond to external stimuli, and withstand various stresses (such as changes in osmolarity). Additionally, chemotaxis and flagella-mediated motility are prerequisites for root colonization. Many genes showed plant-specific dependencies highlighting significant adaptation to different plant species. This work provides a greater understanding of factors promoting rhizosphere fitness and root colonization in plant-beneficial bacteria, facilitating their exploitation for agricultural benefit.

Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino- donor compounds aspartate and glutamate, the reason for the lack of N2 2 fixation in the mutant has remained unclear. During our investigations into the role of AatA, we found that it catalyses an additional transamination reaction between aspartate and pyruvate, forming alanine. This secondary reaction runs at around 60 % of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate-pyruvate transaminase activity R. leguminosarum, , which is common in eukaryotic and many prokaryotic genomes. However, the aspartate- to- pyruvate transaminase reaction is not needed for N2 2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2 2 fixation, rather than biosynthetic transamination to form an amino acid. Hence, the enzyme aspartase, which catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2 2 fixation in pea nodules.

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as anti-toxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP -ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indi-cating a wide distribution of reversible guanine modifications beyond DarTG systems.

The plant common symbiosis signalling (SYM) pathway has shared function between interactions with rhizobia and arbuscular mycorrhizal fungi, the two most important symbiotic interactions between plants and microorganisms that are crucial in plant and agricultural yields. Here, we determine the role of the plant SYM pathway in the structure and abundance of the microbiota in the model legume Medicago truncatula and whether this is controlled by the nitrogen or phosphorus status of the plant. We show that SYM mutants (dmi3) differ substantially from the wild type (WT) in the absolute abundance of the root microbiota, especially under nitrogen limitation. Changes in the structure of the microbiota were less pronounced and depended on both plant genotype and nutrient status. Thus, the SYM pathway has a major impact on microbial abundance in M. truncatula and also subtly alters the composition of the microbiota.

The general stress response (GSR) enables bacteria to sense and overcome a variety of environmental stresses. In alphaproteobacteria, stress-perceiving histidine kinases of the HWE and HisKA_2 families trigger a signaling cascade that leads to phosphorylation of the response regulator PhyR and, consequently, to activation of the GSR σ factor σEcfG. In the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens, PhyR and σEcfG are crucial for tolerance against a variety of stresses under free-living conditions and also for efficient infection of its symbiotic host soybean. However, the molecular players involved in stress perception and activation of the GSR remained largely unknown. In this work, we first showed that a mutant variant of PhyR where the conserved phosphorylatable aspartate residue D194 was replaced by alanine (PhyRD194A) failed to complement the ΔphyR mutant in symbiosis, confirming that PhyR acts as a response regulator. To identify the PhyR-activating kinases in the nitrogen-fixing symbiont, we constructed in-frame deletion mutants lacking single, distinct combinations, or all of the 11 predicted HWE and HisKA_2 kinases, which we named HRXXN histidine kinases HhkA through HhkK. Phenotypic analysis of the mutants and complemented derivatives identified two functionally redundant kinases, HhkA and HhkE, that are required for nodulation competitiveness and during initiation of symbiosis. Using σEcfG-activity reporter strains, we further showed that both HhkA and HhkE activate the GSR in free-living cells exposed to salt and hyperosmotic stress. In conclusion, our data suggest that HhkA and HhkE trigger GSR activation in response to osmotically stressful conditions which B. diazoefficiens encounters during soybean host infection. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

This article comments on: Tian L, Liu L, Xu S, Deng R, Wu P, Jiang H, Wu G, Chen Y. 2022. A d-pinitol transporter, LjPLT11, regulates plant growth and nodule development in Lotus japonicus. Journal of Experimental Botany 73, 351–365.

Rhizobia are a group of bacteria that increase soil nitrogen content through symbiosis with legume plants. The soil and symbiotic host are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the Bradyrhizobium clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens. Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in symbioses with success similar to that of the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the growth defects of extended-hopanoid-deficient mutants can be at least partially compensated for by the physicochemical environment, specifically, by optimal osmotic and divalent cation concentrations. Through biophysical measurements of lipid packing and membrane permeability, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in-culture and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environments within root nodules. To improve the legume-rhizobium symbiosis through either bioengineering or strain selection, it will be important to consider the full life cycle of rhizobia, from soil to symbiosis. IMPORTANCE Rhizobia, such as B. diazoefficiens, play an important role in the nitrogen cycle by making nitrogen gas bioavailable through symbiosis with legume plants. As climate change threatens soil health, this symbiosis has received increased attention as a more sustainable source of soil nitrogen than the energy-intensive Haber-Bosch process. Efforts to use rhizobia as biofertilizers have been effective; however, long-term integration of rhizobia into the soil community has been less successful. This work represents a small step toward improving the legume-rhizobium symbiosis by identifying a cellular component-hopanoid lipids-that confers robustness to environmental stresses rhizobia are likely to encounter in soil microenvironments as sporadic desiccation and flooding events become more common.

Rhizobia are a phylogenetically diverse group of soil bacteria that engage in mutualistic interactions with legume plants. Although specifics of the symbioses differ between strains and plants, all symbioses ultimately result in the formation of specialized root nodule organs that host the nitrogen-fixing microsymbionts called bacteroids. Inside nodules, bacteroids encounter unique conditions that necessitate the global reprogramming of physiological processes and the rerouting of their metabolism. Decades of research have addressed these questions using genetics, omics approaches, and, more recently, computational modeling. Here, we discuss the common adaptations of rhizobia to the nodule environment that define the core principles of bacteroid functioning. All bacteroids are growth arrested and perform energy-intensive nitrogen fixation fueled by plant-provided C-4-dicarboxylates at nanomolar oxygen levels. At the same time, bacteroids are subject to host control and sanctioning that ultimately determine their fitness and have fundamental importance for the evolution of a stable mutualistic relationship.

The Bradyrhizobium -soybean symbiosis is of great agricultural significance and serves as a model system for fundamental research in bacterium-plant interactions. While detailed molecular insight is available about mutual recognition and early nodule organogenesis, our understanding of the host-imposed conditions and the physiology of infecting rhizobia during the transition from a free-living state in the rhizosphere to endosymbiotic bacteroids is currently limited.

Climate change poses a threat to soil health and agriculture, but the potential effects of climate change on soil bacteria that can help maintain soil health are understudied. Rhizobia are a group of bacteria that increase soil nitrogen content through a symbiosis with legume plants. The soil and symbiosis are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the bradyrhizobia clade, like Bradyrhizobium diazoefficiens , possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens . Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in similarly successful symbioses compared to the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens , the in vitro growth defects of extended hopanoid deficient mutants can be at least partially compensated for by the physicochemical environment, specifically by optimal osmotic and divalent cation concentrations. Through biophysical measurements, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in vitro and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environment within root nodules. To improve the legume-rhizobia symbiosis through either bioengineering or strain selection, it will be important to consider the full lifecycle of rhizobia, from the soil to the symbiosis. Importance Rhizobia, such as B. diazoefficiens , play an important role in the nitrogen cycle by making nitrogen gas bioavailable through symbiosis with legume plants. As climate change threatens soil health, this symbiosis has reentered the spotlight as a more sustainable source of soil nitrogen than the energy-intensive Haber-Bosch process. Efforts to use rhizobia as biofertilizers have been effective; however, long term integration of rhizobia into the soil community has been less successful. This work represents a small step towards improving the legume-rhizobia symbiosis by identifying a cellular component—hopanoid lipids—that confers robustness to environmental stresses rhizobia are likely to encounter in soil microenvironments as sporadic desiccation and flooding events become more common.

Legumes are high in protein and form a valuable part of human diets due to their interaction with symbiotic nitrogen-fixing bacteria known as rhizobia. Plants house rhizobia in specialized root nodules and provide the rhizobia with carbon in return for nitrogen. How-ever, plants usually house multiple rhizobial strains that vary in their fixation ability, so the plant faces an investment dilemma. Plants are known to sanction strains that do not fix nitrogen, but nonfixers are rare in field settings, while intermediate fixers are common. Here, we modeled how plants should respond to an intermediate fixer that was otherwise isogenic and tested model predictions using pea plants. Intermediate fixers were only tolerated when a better strain was not available. In agreement with model predictions, nodules containing the intermediate-fixing strain were large and healthy when the only alternative was a nonfixer, but nodules of the intermediate-fixing strain were small and white when the plant was coinoculated with a more effective strain. The reduction in nod-ule size was preceded by a lower carbon supply to the nodule even before differences in nodule size could be observed. Sanctioned nodules had reduced rates of nitrogen fixation, and in later devel-opmental stages, sanctioned nodules contained fewer viable bacte-ria than nonsanctioned nodules. This indicates that legumes can make conditional decisions, most likely by comparing a local nodule-dependent cue of nitrogen output with a global cue, giving them remarkable control over their symbiotic partners.

By analyzing successive lifestyle stages of a model Rhizobium-legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N-2-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N-2. Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphereprogressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N-2 fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium-legume symbiosis.

The adaptation of rhizobia from the free-living state in soil to the endosymbiotic state comprises several physiological changes in order to cope with the extremely low oxygen availability (microoxia) within nodules. To uncover cellular functions required for bacterial adaptation to microoxia directly at the protein level, we applied a systems biology approach on the key rhizobial model and soybean endosymbiont Bradyrhizobium diazoefficiens USDA 110 (formerly B. japonicum USDA 110). As a first step, the complete genome of B. diazoefficiens 110spc4, the model strain used in most prior functional genomics studies, was sequenced revealing a deletion of a ~202 kb fragment harboring 223 genes and several additional differences, compared to strain USDA 110. Importantly, the deletion strain showed no significantly different phenotype during symbiosis with several host plants, reinforcing the value of previous OMICS studies. We next performed shotgun proteomics and detected 2,900 and 2,826 proteins in oxically and microoxically grown cells, respectively, largely expanding our knowledge about the inventory of rhizobial proteins expressed in microoxia. A set of 62 proteins was significantly induced under microoxic conditions, including the two nitrogenase subunits NifDK, the nitrogenase reductase NifH, and several subunits of the high-affinity terminal cbb 3 oxidase (FixNOQP) required for bacterial respiration inside nodules. Integration with the previously defined microoxia-induced transcriptome uncovered a set of 639 genes or proteins uniquely expressed in microoxia. Finally, besides providing proteogenomic evidence for novelties, we also identified proteins with a regulation similar to that of FixK2: transcript levels of these protein-coding genes were significantly induced, while the corresponding protein abundance remained unchanged or was down-regulated. This suggested that, apart from fixK 2, additional B. diazoefficiens genes might be under microoxia-specific post-transcriptional control. This hypothesis was indeed confirmed for several targets (HemA, HemB, and ClpA) by immunoblot analysis.